Fluorescent proteins (FPs) are important reporter molecules for different biomedical applications. In some existing applications, engineered FPs are detected by epi-fluorescence, confocal (microscopy), or reflectance (whole animal) imaging.
Epi-fluorescence, confocal microscopy depends on coherent (non-diffuse) light projected toward and reflected from a specimen. Because microscopy requires substantially coherent light, this technique is only able to image to a small depth (e.g., less than 1 mm) into the specimen. At deeper imaging depths, light is known to become diffuse, rendering microscopy ineffective at the deeper imaging depths.
Reflectance fluorescence imaging has been shown to be useful in detecting and following tumors in vivo, particularly those implanted near the surface or in surgically exposed organs. However, reflectance fluorescence imaging has inherent limitations, since obtained images are a superposition of fluorescence signals from multiple depths, which tends to result in blurred images. Furthermore, reflectance fluorescence imaging is not tomographic and does not retrieve depth information or allow absolute quantification of fluorescence activity. This is due in part to non-linear light attenuation and propagation in biological tissues, which limits the applicability of reflectance fluorescence imaging to semi-quantitative imaging at depths of only a few millimeters.
Imaging optical signatures deeper in tissues often requires the application of advanced light excitation and light detection apparatus and techniques and the use of tomographic principles for combining data acquired at different projections. Advances in imaging with diffracting light sources have resulted in several studies investigating tissue using intrinsically or extrinsically administered optical contrast. In particular, diffuse optical tomography (DOT) is a technique that can provide a tomographic image associated with a diffuse media in the presence of absorption and scattering in the diffuse media. For example, DOT has been applied to cerebral hemodynamic imaging and imaging of breast tissue. One exemplary DOT method and system is described, for example, in international patent application PCT/US04/03229, by Vasilis Ntziachristos and Jorge Ripoll, entitled “Method and System for Free Space Optical Tomography of Diffuse Media,” filed Feb. 5, 2004, which application is assigned to the assignee of the present invention.
It has been shown that light with wavelengths in the near-infrared range can propagate through tissue for distances on the order of several centimeters because of low tissue absorption in the so-called “near-infrared window.” The near-infrared (NIR) window has enabled the development of NIR fluorescence techniques to visualize specific biochemical events inside living specimens.
A variety of related methods for processing NIR fluorescent signals have also been developed. In particular, development of appropriate imaging systems has enabled the application of Fluorescence Molecular Tomography (FMT), a technique that resolves molecular signatures in deep tissues using NIR fluorescent probes or markers. FMT used for in vivo three-dimensional imaging of enzymatic activity in deep-seated tumors has been demonstrated.
A common assumption in conventional NIR optical tomography is that propagation in a diffuse media has high scattering but relatively low absorption, as provided by the NIR window. This assumption has allowed derivation of a “diffusion equation” associated with a “transport equation,” by means of a “diffusion approximation,” which provides an effective tool for modeling NIR photon propagation in tissues. The transport equation is described, for example, in K. M. Case and P. F. Zweifel, “Linear Transport Theory,” Addison-Wesley, Mass., (1967) and in K. Furutsu and Y. Yamada, “Diffusion Approximation for a Dissipative Random Medium and the Applications,” Phys. Rev. E 50, 3634 (1994).
As is known, all currently available fluorescent proteins utilize excitation light having a wavelength in the visible range. Moreover, conventional fluorescent proteins emit visible fluorescent light when excited. Tomographic imaging using visible light, as provided by the conventional fluorescent proteins, is complicated by a relatively high absorption of visible light propagating in biological tissue, which results in significant attenuation. With high absorption, (e.g., for visible light) the conventional diffusion approximation described above is not valid.
Other, more advanced solutions (other than the above-described diffusion approximation) to the transport equation have been generated and applied to NIR optical tomography. The advanced solutions overcome the inadequacy of the above-mentioned diffusion approximation. However the advanced solutions to the transport equation are generally computationally expensive and become impractical for tomographic systems having a large number of excitation light sources, resulting in large data sets.
In order to provide a plurality of images necessary for tomography, many conventional optical tomography systems use an optical switch as part of a light source assembly in order to use a single light element to project at a variety of angles or positions relative to a specimen. It is known that the optical switch generates energy losses. Furthermore, many optical tomography systems use a CCD camera at room temperature or at moderate cooling to collect light. It is known that a room temperature or moderately cooled CCD camera exhibits a relatively high level of dark (thermal) noise, which tends to limit the quality of resulting optical tomography images.